5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. To address the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, we have integrated process and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The process concept presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive and efficient platform for further development, thereby laying the groundwork for a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.

Saturated N-heterocycles constitute a vital scaffold for pharmaceutical chemistry, but are challenging to access synthetically, particularly in asymmetric mode. Here we demonstrate how imine reductases can achieve annulation through tandem inter and intramolecular reductive amination processes. Imine reductases were used in combination with further enzymes to access un-substituted, α-substituted and α,α’-disubstituted N-heterocycles from simple starting materials, in one pot and under benign conditions. The work was exemplified in regard to product scope and a new route to the valuable natural product nicotine was demonstrated.

The enzymatic atroposelective synthesis of biaryl compounds is relatively rare, despite considerable attention received by biocatalysis in the academic and industrial sectors. Imine reductases (IREDs) are an important class of enzymes that have been applied in the asymmetric synthesis of chiral amine building blocks. In this study, two IREDs (IR140 and IR189) were identified to catalyze the efficient desymmetrization of biaryls utilizing various amine donors. Further protein engineering enabled the identification of variants (IR189 M8–M9 and IR189 M13–M14) that are able to catalyze the formation of both ( R ) and ( S ) atropisomers in excellent yields and atroposelectivities (24 examples, up to 99 % ee and yield). The absolute configuration and rotational barriers were confirmed, and the reactions were readily scaled up to allow isolation of the atropisomeric product in 99 % ee and 82 % yield. The optically pure biaryl amines were further derivatized into various synthetically useful atropisomers. To shed light on the molecular recognition mechanisms, molecular dynamics (MD) simulations were performed, offering plausible explanations for the improved atroposelectivity and enzymatic activity. The current strategy expands the scope of the IRED-catalyzed synthesis of axially chiral biaryl amines, contributing significantly to the field of atroposelective biocatalysis.

Nucleosides functionalized at the 2 '-position play a crucial role in therapeutics, serving as both small-molecule drugs and modifications in therapeutic oligonucleotides. However, the synthesis of these molecules often presents substantial synthetic challenges. Here we present an approach to the synthesis of 2 '-functionalized nucleosides based on enzymes from the purine nucleoside salvage pathway. Initially, active-site variants of deoxyribose-5-phosphate aldolase were generated for the highly stereoselective synthesis of d-ribose-5-phosphate analogues with a broad range of functional groups at the 2-position. Thereafter, these 2-modified pentose phosphates were converted into 2 '-modified purine analogues by construction of one-pot multienzyme cascade reactions, leading to the synthesis of guanosine (2 '-OH) and adenosine (2 '-OH, 2 '-Me, 2 '-F) analogues. This cascade allows for the control of the 2 '-functional group alongside 2-stereochemistry. Our findings demonstrate the capability of these biocatalytic cascades to efficiently generate 2 '-functionalized nucleosides, starting from simple starting materials. The chemical synthesis of nucleoside analogues with modifications at the 2-position often requires multiple steps and the extensive use of protecting groups. Now, biocatalytic cascades are reported for the synthesis of 2-functionalized sugars and 2 '-functionalized nucleosides, using enzymes derived from those of the purine nucleoside salvage pathway.

We report the development of an engineered aminotransferase for the synthesis of a key chiral intermediate of the anti-HIV drug Lenacapavir. Due to the sterically demanding nature of the ketone substrate, a substrate walking approach was adopted during directed evolution to unlock desired aminotransferase activity starting from a parent template (TA25) with no observable activity for the target reaction. Introduction of 6 mutations into TA25 over 4 rounds of directed evolution led to the development of an engineered aminotransferase that affords the target chiral amine product with 90% conversion and >99% e.e. in favor of the desired S-enantiomer. The enzyme serves as a valuable template for the development of an industrial biocatalyst for the manufacture of Lenacapavir

AbstractBiocatalytic dearomatisation offers the advantages of high chemo-, regio- and stereoselectivity over chemical strategies. Mono- and dioxygenases with dearomatising properties are already well-established tools for the synthesis of natural products and beyond. Herein, we review investigations of protein sequence–activity relationships, as well as protein-engineering approaches that have been employed to expand the substrate scope of biocatalysts and achieve product regio- and stereodiversity. Thus, oxidative dearomatising biocatalysts offer an increasingly diverse toolbox for the synthesis of asymmetric, oxidised cyclic scaffolds, as illustrated through selected examples of biocatalytic applications in synthetic routes towards natural products and derivatives thereof. Reductases with dearomatising properties have been less well investigated, so we review recent mechanistic findings which, henceforth, allow for expanding applications of this class of biocatalysts. Additionally, chemoenzymatic strategies have been developed to overcome the limitations of purely biocatalytic or chemical dearomatisation approaches. We highlight examples of those combination strategies for the synthesis of asymmetric privileged motifs.1 Introduction2 Oxidative Biocatalytic Dearomatisation3 Reductive Biocatalytic Dearomatisation4 Chemoenzymatic Dearomatisation5 Conclusion

We report the development of an engineered P450 monooxygenase that mediates a chemo- and stereo-selective alkene epoxidation to generate a key chiral precursor of the anti-tuberculosis drug delamanid. Screening of an in-house P450 monooxygenase panel led to the identification of a BM3 variant, containing five mutations, with activity for the target transformation. Over a single round of laboratory evolution and gene shuffling, three further beneficial mutations were introduced leading to an order of magnitude increase in the de-sired activity, with a total turnover number (TON) of >3000. This newly engineered enzyme generates a chiral epoxide intermediate from an alkene precursor in a single step with 98% e.e. and >97% conversion. Initial efforts to scale the biocatalytic transformation high-lights the potential of the engineered enzyme to provide a more efficient and sustainable route for the manufacture of delamanid.

Nucleosides functionalized at the 2′-position play a crucial role in therapeutics, serving as both small molecule drugs and modifications in therapeutic oligonucleotides. However, the synthesis of these molecules often presents significant synthetic challenges. In this study, we present an approach to the synthesis of 2′-functionalized nucleosides based on enzymes from the purine nucleoside salvage pathway. Initially active-site variants of DERA aldolase were generated for the highly stereoselective synthesis of D-ribose-5-phosphate analogs with a broad range of functional groups at the 2-position. Thereafter these 2-modified pentose phosphates were converted into 2′-modified purine analogs by construction of one-pot multi-enzyme cascade reactions, leading to the synthesis of guanosine (2′-OH) and adenosine (2′-OH, 2′-Me, 2′-F) analogues. Our findings demonstrate the capability of these biocatalytic cascades to efficiently generate 2′ functionalized nucleosides, starting from simple starting materials.

Biocatalysis is emerging as an attractive option for manufacturing pharmaceuticals. However, the identification of enzymes for target transformations of interest requires major screening efforts. Here we report a structure-based computational workflow to prioritize protein sequences by a score based on predicted activities on substrates, thereby reducing resource intensive laboratory-based biocatalyst screening. We selected imine reductases (IREDs) as a class of biocatalysts to illustrate the application of the computational workflow termed IREDFisher. Validation by using published data showed that IREDFisher can retrieve the best enzymes and increase the hit rate by identifying the top 20 ranked sequences. The power of IREDFisher is confirmed by computationally screening 1,400 sequences to identify suitable biocatalysts for five selected reductive amination reactions. IREDFisher is available as a user-friendly web interface that will enable rapid identification of biocatalysts for applications in synthesis and directed evolution studies with minimal time and resource expenditure.

Chiral amines are crucial precursors of various pharmaceutical drugs, fine chemicals, and bioactive molecules. Amine dehydrogenases (AmDHs) have attracted increasing attention for chiral-amine synthesis, with great potentials to overcome the shortcomings of conventional chemical methods. However, the narrow substrate scope and expensive cofactors required can be quite challenging for the AmDHs. A number of attempts have been made to improve AmDHs and their reaction systems. The available AmDHs are mainly obtained by the discovery of the natural AmDHs and the engineering of amino acid dehydrogenases. In this review, recent advances in the structures, mechanisms, and mutation studies of AmDHs are discussed in detail, facilitating the engineering of AmDHs more efficiently. Besides, various enzyme-coupled and immobilization strategies have been designed to optimize the AmDH-catalyzed reaction systems, not only achieving the cofactor regeneration but also enhancing the productivity. Future research directions for improving the performance and reaction system of AmDHs are evaluated.

The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of a cytidine aminotransferase for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >100,000 turnovers in less than 30 minutes, operates at 180 g/L substrate loading and benefits from in situ crystallization of the N-hydroxy-cytidine product (>90% yield), which can be converted to Molnupiravir by a selective 5’-acylation using Novozym® 435.

Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deaminase (CD). The wild-type enzyme operates efficiently at high sustrate loadings and hydroxylamine concentrations to favor N-hydroxy-cytidine formation over uridine. Although the wild-type enzyme demonstrated good activity, we were able to further enhance the ratio of N-hydroxycytidine to uridine produced through directed evolution of CD. In particular, a T123G mutation close to the active site dramatically reduces cytidine hydrolysis activity whilst preserving desired amination activty. The approach reported provides a new route to a key intermediate for the COVID-19 experimental drug Molnupiravir 1.

The reductive aminase from Aspergillus oryzae (AspRedAm) was combined with a single alcohol dehydrogenase (either metagenomic ADH-150, an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-neutral cascade for the biocatalytic alkylation of amines using primary and secondary alcohols. Aliphatic and aromatic secondary amines were obtained in up to 99% conversion, as well as chiral amines directly from the racemic alcohol precursors in up to > 97% ee, releasing water as the only byproduct.